Summary Faithful duplication of eukaryotic chromosomes during cell division requires the accurate replication of both chromosomal DNA and its associated chromatin states. However, DNA replication results in the disassembly of nucleosomes ahead of replication forks and thus poses a significant challenge to the integrity of chromatin. Chromatin restoration following DNA replication is initiated by pathways that recycle parental histones or that assemble nucleosomes de novo from newly synthesized histones. As parental histones carry epigenetic modifications, the recycling of parental histones into the daughter genomes is of particular significance for the transmission of epigenetic information across generations. The redundancy of cellular chromatin assembly pathways and the lack of functional biochemical assay systems has limited the mechanistic study of replication-coupled chromatin assembly. We have recently reconstituted the eukaryotic DNA replication reaction using purified budding yeast proteins and yeast origin-containing plasmid templates. The system recapitulates key features of cellular DNA replication, including regulated origin firing and canonical leading and lagging strand synthesis. Parental nucleosomes are also efficiently recycled on the newly replicated DNA by core replisomes in this system. However, extended nucleosome arrays are not reestablished, presumably due to the absence of the de novo nucleosome assembly pathway. In unpublished results, we have purified the budding yeast orthologs of histone chaperones implicated in replication-coupled chromatin assembly in vivo and demonstrate their ability to coordinately assemble nucleosomes from bound histone H3-H4 and H2A-H2B dimers. Combined, the reconstituted DNA replication and nucleosome assembly reactions put us in position to reconstitute replication-coupled chromatin assembly. Aim 1 focuses on mechanisms of parental histone recycling at the replication fork. We will use our recently reconstituted chromatin replication system to test the template commitment during parental histone transfer, identify histone acceptors within the replisome, and test the role of replisome components in the process. The goal of Aim 2 is to interrogate the mechanism of parental nucleosome segregation during DNA replication in vitro. We will employ a strand-specific sequencing approach to determine the distribution pattern of parental nucleosomes to the leading and lagging arms of the fork and how this distribution is controlled by replisome proteins. We will also assess parental nucleosome positions before and after replication to identify the rules governing nucleosome positioning. In Aim3 we will focus on the reconstitution and characterization of the de novo replication-coupled nucleosome assembly pathway using biochemical, structural, and next generation sequencing approaches.